The present invention refers generally to diagnostic test devices and, more particularly, to a test element for carrying out an immunological sandwich test for the determination of an analyte from a liquid sample comprising a novel control zone.
Diagnostic assays for analyzing liquid samples such as blood, serum, plasma, urine, saliva, sweat, etc., often contain a control mechanism which should ensure the proper performance of the test and its functionality.
A control mechanism which is internal to the test is rather unusual when carrying out diagnostic tests on automated laboratory instruments since the instrument itself usually ensures and/or monitors that the test is carried out properly. In the case of automated laboratory instruments the pipetting volumes or the incubation times and other parameters are for example usually automatically monitored by the automated analyzer. The functionality of the test (with regard to the reagents as well as with regard to the instruments) is usually ensured by additionally determining control samples within a series of measurements.
In a point-of-care (PoC) environment, i.e., in the case of patient sample measurements outside of a specialized laboratory, single determinations are often carried out. The test elements used for this usually contain dried reagents which are redissolved by the sample and thus reactivated for the actual analysis. Immunological test elements such as immunochromatographic test strips or immunological capillary gap test elements have proven to be useful in the PoC environment especially in the field of immunoassays. The immunological test elements are often based on optically detectable reactions that are usually evaluated visually or by means of a measuring instrument (reader). In this case the person carrying out the test has a special responsibility since some functions that are carried out by automated laboratory instruments in the case of wet chemistry tests have to be taken care of by the person carrying out the test (for example pipetting the correct sample volume or adherence to the assay time).
However, the personnel carrying out the test in a PoC environment are usually not specially trained employees. Tests in the PoC field are frequently carried out by doctors, nurses, doctor's assistants, pharmacists or by the patients themselves. In contrast, in specialized laboratories (e.g., central laboratories in hospitals, large laboratories) the assays are carried out by specially trained technical assistants (e.g., MTA).
Therefore, test suppliers have developed so-called on-board-controls for these special “decentral” or PoC applications which respond to major handling errors (such as underdosing) when performing the tests and/or to test malfunctions and thus make the user aware of an invalid test result.
In the special case of immunological test elements which function according to the so-called sandwich principle, “on-board controls” are usually implemented in the form of control lines or zones (both terms are used synonymously in the following).
These control lines or zones serve in the ideal case to ensure that the user recognizes that sufficient sample volume has been applied for correct test performance, that the immunochemical reagents that are impregnated or dried in the test element were still present in a migratable form (i.e., in a form that can be moved in the test element) and that the immunological activity of the reagents that are critical for function was still present within a certain range. Otherwise the control line or the control zone allows incorrect test performance or the unusability of the test element to be recognized and thus helps to prevent false test results from being obtained.
A typical test structure is as follows:                A conjugate zone (also: reagent zone) is usually located in or on the test element between a sample application zone and a detection zone. The sample application zone and conjugate zone can also be identical. The conjugate zone contains an immunological binding partner (typically an antibody or a fragment thereof) that is directed against the analyte and can be redissolved and redetached and thus mobilized by the sample liquid. The binding partner is usually conjugated (so-called conjugate) to a signal-generating enzyme or a so-called direct label (e.g., gold or colored latex particles, fluorescent labels).        The detection zone contains an irreversibly immobilized second independent binding partner directed against the analyte (or a complex formed from the analyte).        If the analyte is present in the sample, it thus accumulates in the detection zone in the form of a so-called sandwich (i.e., an immunological complex of immobilized binding partner, analyte and signal-generating binding partner) and becomes visible, optionally after the addition of further reagents.        In the case of the streptavidin-biotin principle (as a representative of the so-called indirect sandwich principle) the second binding partner is also located in the conjugate zone in the form of a mobilizable biotin conjugate. The detection zone contains irreversibly immobilized streptavidin.        Excess conjugate of binding partner and label or biotin is transported together with the sample into a waste (suction zone, which serves to take up excess sample) that is located “downstream” of the detection zone.        The control zone is usually located between the detection zone and waste.        
The following functional principles for control zones are known to a person skilled in the art and are described with their advantages and disadvantages:
1. Analyte Control Zone
                The control zone located between the detection zone and the waste contains irreversibly immobilized analyte (or an analyte analogue). Excess binding partner-label conjugate is bound to the immobilized analyte or analyte analogue and thus leads to a detectable signal at the control line.        This mechanism is nowadays often covered by polyhapten control zones and therefore the advantages and disadvantages are described in more detail in section 2.        A special disadvantage of the analyte control line is that it is often difficult and laborious to produce the analyte (e.g., an antigen in sufficient quantities).2. Polyhapten Control Zone        The control zone situated between the detection zone and the waste contains a polyhapten directed against the conjugate antibody.        Advantage: In addition to ensuring that the reagents were able to migrate and providing a control for the correct sample volume, the formation of a signal in the control zone also ensures the immunological activity, of the conjugate of analyte binding partner and label.        Disadvantage: At high analyte concentrations in the sample the ability of the binding partner-label conjugate to bind antigen is already saturated before reaching the control zone and the polyhapten in the control zone can capture no more conjugate. Consequently the control mechanism fails.        In this case the user must falsely assume that the test result in the detection zone is invalid.        This is particularly frequently a disadvantage when a wide concentration range has to be covered between the cut-off value of the test (i.e., the lower limit of the measuring range) and the upper concentration range. This is for example the case for pregnancy tests in which the hormone hCG has to be detected in urine at a cut-off of about 10-20 mIU/ml and, on the other hand, hCG values of up to 500,000 mIU/ml are also observed at the end of the first trimester of pregnancy. Such high analyte concentrations result in negative results in the control zone and thus misleadingly simulate an invalid test result in the detection zone.3. Anti-IgG Control Zone (as a Form of an Indirect Control Zone)        The control zone contains an antibody directed against the antibody-label conjugate. This control zone principle is very widespread in pregnancy test strips.        Example: If an analyte-specific, labelled mouse antibody is used, the control zone contains for example a polyclonal antibody directed against mouse antibody (e.g., PAB<mouse Fcγ>S-IgG).        Advantage: The formation of the control line is not influenced by high analyte concentrations in the sample.        Disadvantage: The correct immunological activity of the conjugate is not detected by this mechanism. This has recently led to discussions with the approval authorities e.g., the FDA in the USA, with regard to new approvals of test elements. Another disadvantage is that when interference-eliminating antibodies are used in assays which use blood, the intensity of the signal in the control zone is greatly reduced. Interference-eliminating antibodies are usually used in the test in a 10- to 500-fold excess relative to the analyte-specific conjugate antibody. Unconjugated antibodies of the same species are usually used as interference-eliminating antibodies which are thus also captured by the control line.        If the analyte is absent in the sample this is aggravated by the fact that a large portion of the conjugate already binds in the detection zone and the often low residual amount of conjugate leads to a complete failure of the control zone in the presence of the interference eliminating antibody.4. Indirect Control Line that is not Affected by Interference-Eliminating Antibodies        The control line contains a binding partner which is directed against an “independent label” of the conjugate.        Example: The analyte-specific binding partner of the conjugate is additionally digoxigenylated and captured at the control line by means of an anti-digoxigenin antibody (cf., for example, US-A 2002-0055126).        Advantage: The binding capability of the control line is not reduced by high analyte concentrations. Furthermore, the binding capability of the control line is not reduced by the presence of interference-eliminating antibodies.        Disadvantage: The antibodies of the conjugate have to be additionally specially modified which results in additional effort, cost and time. Furthermore, the control line can again fail when there is a high endogenous content of the selected label.        